Genetic replacement therapy for the lungs of cystic fibrosis (CF) patients has multiple requirements to be effective and safe, including adequate levels of hCFTR mRNA in proximal lung epithelial cells using a vector system that is non-immunogenic and non-toxic. Based on in vitro studies, observations in various strains of CF knockout mice, and correlations with human CF lung disease, there is a consensus that achieving approximately 5-10% of normal levels of hCFTR mRNA may be therapeutic [1-3]. The therapeutic challenge is to achieve this level of wildtype hCFTR mRNA in the context of gene transfer to the CF lung, where the disease process typically results in thick mucus secretions that may reduce gene transfer efficiencies.
DNA nanoparticles comprised of single molecules of DNA compacted with polyethylene glycol (PEG)-substituted lysine peptides enter the apical membrane of lung epithelial cells by binding to cell surface nucleolin, and this complex then efficiently enters the nucleus of these post-mitotic cells [4].  The small size of the DNA nanoparticles is required for entry through the nuclear membrane pore [5], although the size dimension requirements for nuclear uptake in the mouse lung appears somewhat less restrictive than in microinjection studies in cells [5, 6]. DNA nanoparticles are non-toxic and do not stimulate CpG responses in the murine lung [7], and repetitive dosing to the lungs of Balb/c mice does not reduce transgene expression [8]. When applied to the nasal mucosa of CF subjects, no adverse events were associated with the nanoparticles and 8/12 subjects had functional evidence of CFTR chloride channel function, with several in the normal range of nasal potential difference testing [9]. The payload plasmid in this initial phase I trial incorporated a CMV promoter, which rapidly shuts off in the lung [10], since only transient hCFTR expression was desired, thus permitting evaluation of potential nanoparticle toxicity without the added complexity of sustained hCFTR protein effects. In total, these encouraging findings suggest that aerosol delivery of compacted DNA nanoparticles may provide sufficient hCFTR expression to address the lung manifestations of CF.
To address anticipated difficulties in transfecting the human CF lung, optimization of the hCFTR expression plasmid to achieve high level and prolonged mRNA levels is a premium requirement, since the ability to maintain expression in successively transfected airway cells might address the mobile CF mucus environment. Since airway epithelial cells survive approximately 200 days before apoptosis [11], achieving hCFTR transgene mRNA expression for months is the desired objective. To achieve this goal, the DNA nanoparticle plasmid payload must efficiently enter airway cell nuclei and then remain transcriptionally active. Processes that might interfere with long term hCFTR transgene expression include promoter down-regulation [12], CpG methylation and/or heterochromatin formation within the transcriptional cassette [13], and loss of the expression plasmid itself. We report the successful optimization of hCFTR mRNA expression in the lungs of CF knockout mice by employing multiple strategies to affect the level and duration of hCFTR expression. Levels of hCFTR mRNA and protein expression have been significantly enhanced by the development of synthetic hCFTR expression ‘genes’ that optimize codon utilization, deplete CpG islands, and optimize Kozak consensus sequences. Additionally, prolonged duration of hCFTR expression has been achieved with use of a novel expression element derived from a 3′ portion of transcribed sequences of the bovine growth hormone (BGH) gene. In combination, these hCFTR expression plasmids, delivered as compacted DNA nanoparticles to the lungs of CF knockout mice, achieve significant expression levels for multiple months, thereby addressing optimization goals for aerosol lung testing in CF subjects.